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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5472_Библиотеки_им_академика_М_И_Перельмана.pdf
Proteins and Proteomics
45
a specific three-dimensional shape that is unique to it. The conformation adopted is dependent on
the polypeptide’s amino acid sequence, and this conformation is largely stabilized by multiple and
weak noncovalent interactions. A structure protein cannot be totally predicted from its amino acid
sequence. Its conformation can, however, be determined by techniques such as X-ray diffraction
and nuclear magnetic resonance spectroscopy.
The shape into which a protein naturally folds is known as its native conformation. Although
many proteins can fold unassisted through the chemical properties of their amino acids, others
require the aid of molecular chaperones (proteins) to fold into their native states.
The proteins are classified broadly into the following:
1. Simple proteins
2. Conjugated proteins
Simple Proteins
These consist exclusively of polypeptide chain(s) with no additional chemical components.
Conjugated Proteins
In addition to their polypeptide moieties, these proteins contain one or more nonpolypeptide
constituents known as prosthetic group. Commonly occurring prosthetic groups in association with
proteins include carbohydrates, vitamin derivatives, phosphate groups and metal ions.
The total number of amino acids, molecular mass, number of polypeptides present and functions
of some protein are given in Table 4.2.
Primary Structure of Proteins
Polypeptides are linear and unbranched polymers potentially containing up to 20 different
monomer types linked together in a precise predefined sequence (Fig. 4.2). The primary structure
Table 4.2 Structure, function and uses of important proteins
Proteins
Total no. of
amino acids
Molecular
mass
No. of
polypeptide
chains
Function/use
Insulin (human) 51 5800 2 Complex nature and biological regulation
of blood glucose level
Lysozyme (egg) 129 13,900 1 Used for degrading peptidoglycan in bac-
terial cell walls
Human erythropoietin 165 36,000 3 Stimulates red blood cell production
Chymotrypsin 241 21,600 3 Digestive proteolytic enzyme
Haemoglobin 574 64,500 4 Used for gas transport
Tumour necrosis 471 52,000 3 Used as mediator of inflammation and
immunity
Hexokinase 800 1,02,000 2 Used for phosphorylating selected mono-
saccharides
Textbook of Pharmaceutical Biotechnology
46
Figure 4.2 Chemical structures of amino acids found in proteins. (Continued...)
of a polypeptide is related to its amino acid sequence, along with the exact positioning of any
disulphide bonds present.
Proteins and Proteomics
47
Figure 4.2 Chemical structures of amino acids found in proteins.
Secondary Structure of Proteins
These are regular repeating local structures stabilized by hydrogen bonds. The most common
examples are the a-helix, b-sheet and turns. Secondary structures are local. Therefore, many
regions of different secondary structures can be present in the same protein molecule. The a-helix
contains 3,6-amino acid residues in a full turn, which is approximately 0.56 nm in length along
the long axis of the helix. The participating amino acid side chains protrude outwards from the
helical backbone. Amino acids that are most conducive with a-helix formation are alanine, leucine,
methionine and glutamate. The helical structure is stabilized by hydrogen bonding, with every
backbone C=O group forming a hydrogen bond with N–H group, four residues ahead of it in the
helix. The stretches of a-helix found in globular (i.e. tightly folded and approximately spherical)
polypeptides can vary in length from a single helical turn to greater than 10 consecutive helical
turns (Figs. 4.3 and 4.4).
Textbook of Pharmaceutical Biotechnology
48
Tertiary Structure of Proteins
The term tertiary structure is often used synonymously with the term fold. It is generally stabilized
by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through
salt bridges, hydrogen bonds, disulphide bonds and even posttranslational modifications. The
tertiary structure controls the basic function of the protein. A polypeptide’s tertiary structure refers
to its exact three-dimensional structure, relating the relative positioning in space of all constituent
atoms of polypeptide to each other. The tertiary structure of small polypeptides (approximately
200 amino acid residues or less) usually forms a single discrete structural unit. However, when the
Figure 4.4 Structure of b-sheet.
Figure 4.3 Ball-and-stick and ribbon representations of an a-helix.
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Proteins and Proteomics
49
three-dimensional structure of many larger polypeptides is examined, the presence of two or more
structural subunits within the polypeptide becomes apparent.
Quaternary Structure of Proteins
This structure is formed by several protein molecules (polypeptide chains), usually called protein
subunits that functions as a single protein complex.
Methods of Studying Proteins
A particular protein can be studied by developing an antibody that is specific to that modification.
For example, there are antibodies (pan antibodies) that recognize only certain proteins when they are
tyrosine phosphorylated. These can be used to determine the set of proteins that have undergone the
modification of interest. The posttranslational modification of interest can be determined by subjecting
a complex mixture of proteins to two-dimensional gel electrophoresis (2-DE). In this technique,
proteins are electrophoresed first in one direction and then in another. This allows small differences in
a protein to be visualized by separating a modified protein from its unmodified form.
Recently, another approach called PROTOMAP has been developed that combines SDS-
PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) with shotgun proteomics.
This enables the detection of changes in gel migration, such as those caused by proteolysis or
posttranslational modification. To an important degree, antibodies to particular proteins or to their
modified forms have been used as common analytical tools in biochemistry and cell biology studies.
The technique such as ELISA can be used for quantitative determination of protein. For proteomic
study, more recent techniques such as matrix-assisted laser desorption/ionization (MALDI) are
employed for rapid determination of proteins in particular mixtures.
Most proteins function in collaboration with other proteins. One goal of proteomics is to
identify which proteins interact. Several methods are available to probe protein–protein interactions.
The traditional method is yeast two-hybrid analysis. New methods are protein microarrays,
immunoaffinity chromatography, followed by mass spectrometry (MS), dual polarization
interferometry and experimental methods, such as phage display and computational methods.
The laboratory should have access for undertaking the following assays:
1. A rapid, reliable assay for the target protein
2. Purity determination
3. Total protein determination
4. Assay for impurities that must be removed
The importance of a reliable assay for the target protein cannot be overemphasized. The purity
of the target protein is most often estimated by SDS-PAGE, capillary electrophoresis, reversed
phase chromatography or mass spectrometry. For large-scale protein purification, assay of target
protein and critical impurities is often essential.
Textbook of Pharmaceutical Biotechnology
50
PROTEOMICS
Proteomics is a systematic research approach aiming to provide the global characterization of protein
expression and function under given conditions. Proteomics techniques have been widely adopted in
the areas of biology and medicine. Like any new technology, when it is first introduced, proteomics
has also been promoted with much hope and promise. Proteomic technology is used to elucidate
protein changes between healthy and diseased states. The application of proteomic technique to the
field of medicine is slowly transforming the way biomarker discovery is conducted.
Clinical Applications of Proteomics
In medical research, it has become increasingly apparent that cellular processes, particularly in
diseases, are determined by multiple proteins. Hence, it is important not to focus on one single gene
product (one protein) but to study the complete set of gene products (the proteome). In this way,
the multifactorial relations underlying certain diseases may be unravelled potentially identifying
therapeutical targets. For many diseases, characterization of the functional proteome is crucial for
elucidating alterations in protein expression and modifications.
Proteomics in Lab Diagnosis
The rapid spread of proteomic technology—which principally consists of 2-DE with in-gel
protein digestion of protein spots and identification by MS—has provided an explosive amount of
diagnostic results. Proteomics plays an important role in the translation of genomics to clinically
useful applications, especially in the areas of diagnostics and prognostics. The emergences of new
techniques such as protein and peptide microarrays are useful in laboratory diagnosis. The role
of proteomics in the development of new laboratory diagnostics and the implications for routine
diagnosis and monitoring of diseases are much evident today. The application of high-resolution
mass spectrometers and other quantitative proteomic methodologies to laboratory diagnosis is a
reality today.
In the coming decades, proteomic technologies shall broaden our understanding of the
underlying mechanisms and further our ability to diagnose, prognosticate and treat many diseases.
Proteomics in the Discovery of Protein Biomarkers
Proteomics, with its high-throughput and unbiased approach to the analysis of variations in protein
expression patterns (actual phenotypic expression of genetic variation), promises to be the most
suitable platform for biomarker discovery. Biomarkers of drug efficacy and toxicity are key factors
in the drug development process. The success of biomarker discovery effort depends upon the
quality of samples analysed, the ability to generate quantitative information on relative protein
levels and the ability to readily interpret the data generated. Proteomic technology is widely used
in biomarker discovery and pathogenic studies. Mass spectral-based proteomic technologies are
ideally suited for the discovery of protein biomarkers in the absence of any prior knowledge of
quantitative changes in protein levels.
Proteins and Proteomics
51
Proteomics in Neurology
In neurology and neuroscience field, proteomics has applications in neurotoxicology and
neurometabolism, as well as in the determination of specific proteomic aspects of individual
brain areas and body fluids in neurodegeneration. Investigations of brain protein groups in
neurodegeneration—such as enzymes, cytoskeleton proteins, chaperones, synaptosomal proteins
and antioxidant proteins—are in progress as phenotype-related proteomics.
Proteomics in Diagnosis
In the diagnosis and treatment of kidney disease, a priority is the identification of disease-associated
biomarkers. Combining important analytical techniques of 2-DE with more sophisticated techniques
such as MS has enabled considerable progress in cataloguing and quantifying proteins. This type
of measurement has been made possible in urine and various kidney tissue compartments in both
normal and diseased physiological states.
Given the dynamic range and complexity of proteomics, completely defining the proteome in
various biological compartments (e.g. tissues, serum and urine) in both health and diseases remains
a major challenge.
Proteomics in the Study of Tumour Metastasis
The molecular and cellular mechanisms underlying tumour metastasis, which is the dominant cause
of death in cancer patients, are still difficult to describe. The identification of protein molecules
with their expressions correlated to metastatic process is helpful in understanding the metastatic
mechanisms and thus, facilitating the development of strategies for the therapeutic interventions and
clinical management of cancer. The combination of proteomics with other experimental approaches
in cell biology, biochemistry, chemistry and molecular genetics together with the development of
new technologies in existing methodologies shall have focused applications in studying cancer
metastasis.
Proteomics in Neurotrauma
Proteomics provides a valuable approach to evaluate the posttraumatic central nervous system
(CNS). Although a comprehensive assessment of all methods for protein analysis cannot be
provided, some of the newer proteomic technologies that have propelled this field into the limelight
and that are available to most researchers in neurotrauma are described.
Neurotrauma results in complex alterations to the biological systems within the nervous system,
and these changes evolve over time. Exploration of the ‘new nervous system’, which follows injury,
requires methods that can both fully assess and simplify this complexity. Proteomics is likely to
be very useful for developing diagnostic predictors after CNS injury and for mapping changes in
proteins after injury in order to identify new therapeutic targets.
Proteomics in Urological Cancer Research
The comparison of the proteins present in a diseased tissue sample with the proteins present in a
normal tissue sample is possible with proteomics analysis. Proteins that are altered either qualitatively
Textbook of Pharmaceutical Biotechnology
52
or quantitatively between the normal and diseased sample are likely to be associated with the
disease process. These proteins after identification can serve the purpose of diagnostic markers for
early detection of the disease. Alternatively, they can be prognostic markers to predict the outcome
of the disease, or they may be used as drug targets for the development of new therapeutic agents.
Proteomics in the Study and Treatment of Autoimmune System
Proteomics provides a powerful tool to characterize autoreactive B cell responses in diseases,
including rheumatoid arthritis, autoimmune diabetes, multiple sclerosis and systemic lupus
erythematosus. Autoantibody profiling may be useful in classification of individual patients and
subsets of patients based on their ‘autoantibody fingerprint’, discovery and characterization of
candidate autoantigens, examination of epitope spreading and antibody isotype usage in addition to
tailoring antigen-specific therapy.
Proteomics in Diabetes Research
The techniques involved in proteomics allow the global screening of complex samples of proteins
and provide qualitative and quantitative evidences of altered protein expression. This lends to
the investigations of the molecular mechanisms underpinning disease processes and the effects
of treatment. When proteins undergo nongenetically determined alterations, such as alternative
splicing or posttranslational modifications (phosphorylation or glycosylation), it may affect their
function. Although abnormalities in splicing or posttranslational modifications may cause a disease
process, they can also be a consequence. Diabetes has high blood glucose, which glycosylates
hundreds or thousands of proteins, including HbA
1c
, which is used to monitor diabetes.
Proteomics in Cardiovascular Research
The analysis undertaken by proteomic technologies at the organ, subcellular and molecular
levels have revealed dynamic, complex and subtle intracellular processes associated with heart
and vascular diseases. The power and flexibility of proteomic analyses, which facilitate protein
separation, identification and characterization, should hasten our understanding of these processes
at the protein level. Evolution of proteomic techniques has permitted detailed investigations into
molecular mechanisms underlying cardiovascular diseases and facilitating identification not only
of modified proteins but also of the nature of their modifications.
Proteomics in Investigating Plants
Proteomics offers novel techniques to study all facets of protein structure and functions in plants.
The application of proteomics in plant pathology is more evident with techniques such as 2-DE and
MS being used to characterize cellular and extracellular virulence and pathogenicity factors. It is
useful to identify changes in protein levels in host plant upon infection by pathogenic organism.
The whole genome sequences are now available for plants, such as for Arabidopsis thaliana, Oryza
sativa and others.
Proteins and Proteomics
53
QUESTIONS
1. What is protein engineering?
2. Write in brief about the denaturation of proteins.
3. Write a note on differences between protein biochemistry and proteomics.
4. Differentiate primary and secondary structures of proteins.
5. Define proteomics and give its applications.
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